Captured image evaluation apparatus, captured image evaluation method, and captured image evaluation program

ABSTRACT

Provided are a captured image evaluation apparatus, a captured image evaluation method, and a captured image evaluation program capable of evaluating a thickness and a density of stacked cultured cells in a short imaging time. The captured image evaluation apparatus includes: an image acquisition section  52  that acquires captured images obtained by imaging a subject under a condition in which a numerical aperture of an objective lens is changed; a thickness estimation section  53  that estimates a thickness of the subject on the basis of a low NA captured image obtained under a condition in which the numerical aperture of the objective lens is relatively small; and a density estimation section  54  that estimates a density of the subject on the basis of a high NA captured image obtained under a condition in which the numerical aperture of the objective lens is relatively large.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of PCT International Application No.PCT/JP2017/026745 filed on Jul. 25, 2017, which claims priority under 35U.S.C § 119(a) to Patent Application No. 2016-189793 filed in Japan onSep. 28, 2016, all of which are hereby expressly incorporated byreference into the present application.

BACKGROUND OF THE INVENTION 1. Field of the Invention

The present invention relates to a captured image evaluation apparatus,a captured image evaluation method, and a non-transitory computerrecording medium storing a captured image evaluation program forperforming estimation of a thickness and a density of a subject based ona captured image of the subject.

2. Description of the Related Art

Multi potential stem cells such as embryonic stem (ES) cells or inducedpluripotent stem (iPS) cells are capable of being divided into cells ofvarious tissues, and attract attention as applications to regenerativemedicine, drug development, disease solution, or the like.

It is known that cells are stacked in three-dimensions as they grow up.It is important to know a thickness and a density (degree ofconcentration of cells) of cultured cells that are stacked in a casewhere the cultured cells are used in a field of regenerative medicineand drug discovery.

As a method for evaluating a state of the cultured cells, in the relatedart, a method for imaging cultured cells using a microscope such as aphase difference microscope and recognizing features of the capturedimage to evaluate a cell culture state has been proposed.

For example, in order to evaluate a state of cultured cells that arestacked as described above, JP2016-021915A proposes a method for movinga focal position of a detection optical system in a direction where thecultured cells are stacked to image a plurality of sectional imageshaving different distances in the stacked direction from a surface onwhich the cultured cells are provided.

SUMMARY OF THE INVENTION

However, in capturing the plurality of sectional images while changingthe focal position, as in the method disclosed in JP2016-021915A, thereis a problem in that the number of times of capturing of the sectionalimages becomes large, and thus, an imaging time becomes long.

Further, since it is necessary to perform an analysis process byintegrating multiple sectional images, a load on the analysis processalso becomes large.

In consideration of the above-mentioned problems, an object of theinvention is to provide a captured image evaluation apparatus, acaptured image evaluation method, and a non-transitory computerrecording medium storing a captured image evaluation program capable ofevaluating a thickness and a density of stacked cultured cells in ashort imaging time.

According to an aspect of the invention, there is provided a capturedimage evaluation apparatus comprising: an image acquisition section thatacquires a plurality of captured images obtained by imaging a subjectunder a condition in which a numerical aperture of an objective lens ischanged; a thickness estimation section that estimates a thickness ofthe subject on the basis of a low NA captured image obtained under acondition in which the numerical aperture of the objective lens isrelatively small, among the plurality of captured images; and a densityestimation section that estimates a density of the subject on the basisof a high NA captured image obtained under a condition in which thenumerical aperture of the objective lens is relatively large, among theplurality of captured images.

In the captured image evaluation apparatus according to the aspect ofthe invention, the thickness estimation section may acquire a brightnessdistribution of the low NA captured image, and may estimate thethickness of the subject on the basis of the brightness distribution.

In the captured image evaluation apparatus according to the aspect ofthe invention, the thickness estimation section may include a table inwhich a brightness of the low NA captured image and the thickness of thesubject are associated with each other.

In the captured image evaluation apparatus according to the aspect ofthe invention, the density estimation section may acquire a brightnessdistribution of the high NA captured image, and may estimate the densityof the subject on the basis of the brightness distribution.

In the captured image evaluation apparatus according to the aspect ofthe invention, the density estimation section may estimate the densityof the subject on the basis of a shape of the brightness distribution.

In the captured image evaluation apparatus according to the aspect ofthe invention, the density estimation section may calculate a peakincluded in the brightness distribution, and calculates the number ofpeaks per unit area to estimate the density of the subject.

In the captured image evaluation apparatus according to the aspect ofthe invention, the density estimation section may approximate thebrightness distribution using a Gaussian function to calculate the peak.

In the captured image evaluation apparatus according to the aspect ofthe invention, the low NA captured image may be a captured imageobtained at a relatively low magnification, and the high NA capturedimage may be a captured image obtained at a relatively highmagnification.

In the captured image evaluation apparatus according to the aspect ofthe invention, the low NA captured image may be a captured imageobtained by illumination light of a relatively long wavelength, and thehigh NA captured image may be a captured image obtained by illuminationlight of a relatively short wavelength.

In the captured image evaluation apparatus according to the aspect ofthe invention, the low NA captured image may be a captured imageobtained using an aperture stop having a relatively small aperture, andthe high NA captured image may be a captured image obtained using anaperture stop having a relatively large aperture.

The captured image evaluation apparatus according to the aspect of theinvention may further comprise: an output section that outputs thethickness of the subject estimated by the thickness estimation sectionand the density of the subject estimated by the density estimationsection.

According to another aspect of the invention, there is provided acaptured image evaluation method comprising: acquiring a plurality ofcaptured images obtained by imaging a subject under a condition in whicha numerical aperture of an objective lens is changed; estimating athickness of the subject on the basis of a low NA captured imageobtained under a condition in which the numerical aperture of theobjective lens is relatively small, among the plurality of capturedimages; and estimating a density of the subject on the basis of a highNA captured image obtained under a condition in which the numericalaperture of the objective lens is relatively large, among the pluralityof captured images.

According to still another aspect of the invention, there is provided anon-transitory computer recording medium storing a captured imageevaluation program that causes a computer to function as: an imageacquisition section that acquires a plurality of captured imagesobtained by imaging a subject under a condition in which a numericalaperture of an objective lens is changed; a thickness estimation sectionthat estimates a thickness of the subject on the basis of a low NAcaptured image obtained under a condition in which the numericalaperture of the objective lens is relatively small, among the pluralityof captured images; and a density estimation section that estimates adensity of the subject on the basis of a high NA captured image obtainedunder a condition in which the numerical aperture of the objective lensis relatively large, among the plurality of captured images.

According to the captured image evaluation apparatus, the captured imageevaluation method, and the captured image evaluation program of theinvention, captured images obtained by imaging a subject under acondition in which a numerical aperture of an objective lens is changedare acquired; a thickness of the subject on the basis of a low NAcaptured image obtained under a condition in which the numericalaperture of the objective lens is relatively small is estimated; and adensity of the subject on the basis of a high NA captured image obtainedunder a condition in which the numerical aperture of the objective lensis relatively large is estimated. Accordingly, since it is sufficientthat two times of imaging for the low NA captured image and the high NAcaptured image is performed, it is possible to evaluate the thicknessand the density of the subject in a short imaging time.

Here, the reason why the low NA captured image is used in a case wherethe thickness of the subject is estimated is because a captured imageincluding a large amount of rectilinear propagation components of lightpassed through the subject, in which the thickness of the subject isconsidered, is obtained in a case where the numerical aperture of theobjective lens is small. Further, the reason why the high NA capturedimage is used in a case where the density of the subject is estimated isthat a captured image including a large amount of scattering componentsof light passed through the subject, in which the density of the subjectis considered, is obtained in a case where the numerical aperture of theobjective lens is large.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram showing a schematic configuration of a microscopeimage evaluation system using an embodiment of a captured imageevaluation apparatus of the invention.

FIG. 2 is a diagram showing an example of a low NA captured image.

FIG. 3 is a diagram showing an example of a brightness distributionacquired on the basis of the low NA captured image.

FIG. 4 is a diagram showing an example of a table showing a relationshipbetween the brightness of the low NA captured image and a thickness of asubject.

FIG. 5 is a diagram showing an example of an estimation result of thethickness of the subject.

FIG. 6 is a diagram showing an example of a low NA captured image.

FIG. 7 is a diagram showing an example of a brightness distributionacquired on the basis of the low NA captured image.

FIG. 8 is a diagram showing a result obtained by approximating thebrightness distribution shown in FIG. 7 using a Gaussian function.

FIG. 9 is a diagram showing an example of a high NA captured imageincluding an image of rubbish.

FIG. 10 is a diagram showing an example of a brightness distributionincluding a peak based on the image of rubbish.

FIG. 11 is a flowchart for illustrating an operation of the microscopeimage evaluation system using the embodiment of the captured imageevaluation apparatus of the invention.

FIG. 12 is a diagram showing a schematic configuration of the microscopeimage evaluation system using another embodiment of the captured imageevaluation apparatus of the invention.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

Hereinafter, a microscope image evaluation system using an embodiment ofa captured image evaluation apparatus and a captured image evaluationmethod will be described in detail with reference to the accompanyingdrawings. FIG. 1 is a diagram showing a schematic configuration of themicroscope image evaluation system of an embodiment of the invention.

The microscope image evaluation system of the embodiment comprises anillumination light emission section 10, an imaging optical system 30, animaging section 40, a microscope control device 50, a display device 80,and an input device 90, as shown in FIG. 1.

In the microscope image evaluation system of the embodiment, a stage 61is provided between the illumination light emission section 10 and theimaging optical system 30, and a culture container 60 is placed andsupported on the stage 61. A culture solution C and a subject S areaccommodated in the culture container 60.

Further, the microscope image evaluation system of this embodimentcomprises a stage driving section 62 for moving the stage 61 in an Xdirection and a Y direction. The X direction and the Y direction aredirections that are orthogonal to each other on a surface parallel to aninstallation surface of the subject S, and a Z direction is a directionthat is orthogonal to the X direction and the Y direction.

In the microscope image evaluation system of this embodiment, theillumination light emission section 10, the imaging optical system 30,the imaging section 40, the stage 61, and the stage driving section 62form a phase difference microscope body, and the microscope controldevice 50 controls the phase difference microscope body. Hereinafter, aspecific configuration of the phase difference microscope body will bedescribed.

The illumination light emission section 10 irradiates the subject Scontained in the culture container 60 with illumination light forso-called phase difference measurement, and in this embodiment,irradiates the culture container 60 with ring-shaped illumination lightas the illumination light for the phase difference measurement.

Specifically, the illumination light emission section 10 according tothe embodiment comprises a white light source 11 that emits white lightfor phase difference measurement, a slit plate 12 that has a ring-shapedslit, to which the white light emitted from the white light source 11 isincident, and that emits ring-shaped illumination light, and a condenserlens 13 to which the ring-shaped illumination light emitted from theslit plate 12 is incident, and that irradiates the subject S with theincident ring-shaped illumination light.

The slit plate 12 is a light screen that screens white light emittedfrom the white light source 11 and is formed with the ring-shaped slitthrough which the white light passes, in which ring-shaped illuminationlight is formed as the white light passes through the slit.

In the culture container 60 provided on the stage 61, a cultured cellgroup (cell colony) are placed as the subject S. As cultured cells,multi potential stem cells such as induced pluripotent stem (iPS) cellsand embryonic stem (ES) cells, cells of nerves, the skin, themyocardium, and the liver, cells of skin extracted from a human body,the retina, the myocardium, blood corpuscles, nerves, and organs, andthe like may be used. As the culture container 60, for example, a wellplate on which a schale and a plurality of wells are arranged may beused.

The imaging optical system 30 forms an image of the subject S inside theculture container 60 on the imaging section 40, and comprises anobjective lens 31, a phase plate 32, an image forming lens 33.

The phase plate 32 is a transparent plate that is formed with a phasering with respect to a wavelength of the ring-shaped illumination light.The size of the slit of the above-mentioned slit plate 12 has aconjugate relationship with the phase ring.

The phase ring has a configuration in which a phase membrane that shiftsa phase of incident light by a ¼ wavelength and a dimmer filter thatdims the incident light are formed in a ring-shape. As direct lightincident to the phase plate 32 passes through the phase ring, its phaseshifts by a ¼ wavelength, and its brightness becomes weak. On the otherhand, diffracted light diffracted by the subject S mostly passes througha portion of the transparent plate of the phase plate 32, and thus, itsphase and brightness are not changed.

The image forming lens 33 is a member to which direct light anddiffracted light passed through the phase plate 32 are incident, whichforms of images of the lights on the imaging section 40.

The image optical system 30 of the embodiment is configured to be ableto change an optical magnification. As a method for changing the opticalmagnification, for example, a method for providing a plurality ofobjective lenses 31 having different magnifications in the imagingoptical system 30 and manually or automatically switching the pluralityof objective lenses 31 may be used. In a case where the magnification ofthe objective lens 31 is changed, the phase plate 32 is also changed inaccordance with the change of the magnification of the objective lens31.

Here, in this embodiment, the thickness and density of the subject S areestimated on the basis of a captured image obtained by imaging thesubject S. Further, in a case where the thickness of the subject S isestimated, a low numerical aperture (NA) captured image obtained byimaging under a condition in which a numerical aperture of the objectivelens 31 is relatively small is used, and in a case where the density ofthe subject S is estimated, a high numerical aperture (NA) capturedimage obtained by imaging under a condition in which a numericalaperture of the objective lens 31 is relatively large is used. Thereason why the low NA captured image is used in a case where thethickness of the subject S is estimated is because a captured imageincluding a large amount of rectilinear propagation components of lightpassed through the subject S, in which the thickness of the subject S isconsidered, is obtained in a case where the numerical aperture of theobjective lens 31 is small. Further, the reason why the high NA capturedimage is used in a case where the density of the subject S is estimatedis because a captured image including a large amount of scatteringcomponents of light passed through the subject S, in which the densityof the subject S is considered, is obtained in a case where thenumerical aperture of the objective lens 31 is large.

In this embodiment, for example, in a case where a command input forperforming estimation of the thickness of the subject S is received froma user, the magnification of the objective lens 31 is automaticallychanged to become relatively low so that the numerical aperture of theobjective lens 31 becomes small. On the other hand, in a case where acommand input for performing estimation of the density of the subject Sis received from the user, the magnification of the objective lens 31 isautomatically changed to become relatively high so that the numericalaperture of the objective lens 31 become large. The relatively lowmagnification may be set to four times, for example, and the relativelyhigh magnification may be set to ten times, for example. Here, themagnification is not limited thereto. The command input from the usermay be received from the input device 90, and the magnification forthickness estimation and the magnification for density estimation may beset in advance.

In this embodiment, the plurality of captured images obtained by imaginga subject under the condition in which the numerical aperture of theobjective lens is changed are not limited to the captured imagesobtained by imaging the subject under the condition in which themagnification of the objective lens is changed as described above, andas described later, may include captured images obtained by imaging thesubject under a condition in which the size of an aperture stop ischanged and captured images obtained by imaging the subject under acondition in which a wavelength of illumination light is changed. Thatis, as long as the numerical aperture of the objective lens is change asa result, a change of different conditions of the optical system may beused, instead of the magnification of the lens.

The imaging section 40 comprises an imaging element that receives lightof an image of the subject S captured by the image forming lens 33 andforms a phase difference image of the subject S. The imaging element mayemploy a charge-coupled device (CCD) image sensor, a complementarymetal-oxide semiconductor (CMOS) image sensor, or the like.

The microscope control device 50 is configured by a computer thatcomprises a central processing unit (CPU), a semiconductor memory, ahard disk, and the like.

The microscope control device 50 controls an entire operation of thephase difference microscope body, and specifically, comprises acontroller 51 that includes the CPU, an image acquisition section 52, athickness estimation section 53, and a density estimation section 54, asshown in FIG. 1. In this embodiment, the microscope control device 50corresponds to the captured image evaluation apparatus according to theembodiment of the invention. In a memory or a hard disk of themicroscope control device 50, an embodiment of the captured imageevaluation program of the invention is installed. As the program isexecuted by the controller 51, the image acquisition section 52, thethickness estimation section 53, and the density estimation section 54perform their functions.

The controller 51 controls an operation of the phase differencemicroscope body, and specifically, controls operations of theillumination light emission section 10, the stage driving section 62,and the imaging section 40, for example.

The controller 51 controls driving of the stage driving section 62, tothereby move the stage 61 in the X direction and the Y direction. As thestage 61 is moved in the X direction and the Y direction, for example,the inside of one cell is scanned by illumination light for phasedifference measurement, and a phase difference image in each of aplurality of imaging regions (fields of view) divided inside one cell isimaged.

The image acquisition section 52 acquires the phase difference image ofthe subject S output from the imaging section 40 as a captured image,and stores the result.

The thickness estimation section 53 estimates the thickness of a cellgroup that is the subject S on the basis of a low NA captured imageobtained at a relatively low magnification. For example, in a case wherea low NA captured image as shown in FIG. 2 is input, the thicknessestimation section 53 acquires a brightness distribution along a singledot chain line shown in FIG. 2. FIG. 3 is a diagram showing an exampleof a brightness distribution. In the thickness estimation section 53, atable indicating a relationship between a brightness of a low NAcaptured image as shown in FIG. 4 and a thickness of the subject S isset in advance. The thickness estimation section 53 calculates acorresponding thickness with reference to the table shown in FIG. 4,with respect to a brightness at each position of the brightnessdistribution shown in FIG. 3, and thus, estimates a distribution ofthicknesses at respective positions of the subject S as shown in FIG. 5.

In the above description, the thickness on the single dot chain lineshown in FIG. 2 is estimated, but the invention is not limited thereto,and a distribution of two-dimensional thicknesses of the subject S maybe estimated by setting line segments that cross the subject S in aplurality of directions and estimating thicknesses on the respectiveline segments. Further, a user may set and input a direction of asegment for estimating a thickness using the input device 90.

Further, the Table shown in FIG. 4 in which the brightness and thethickness are associated with each other may be set for eachmagnification of the objective lens 31. In a case where themagnification of the objective lens 31 is changed, the table may beautomatically changed in accordance with the magnification change.

The density estimation section 54 estimates a density (the degree ofconcentration) of cells of the cell group that is the subject S on thebasis of a high NA captured image obtained at a relatively highmagnification. In a case where a high NA captured image as shown in FIG.6 is input, for example, the density estimation section 54 acquires abrightness distribution along a single dot chain line shown in FIG. 6.FIG. 7 is a diagram showing an example of a brightness distribution.Further, the density estimation section 54 estimates the density ofcells on the basis of the shape of the brightness distribution shown inFIG. 7. Specifically, the density estimation section 54 approximates thebrightness distribution using the Gaussian function. A thick solid lineshown in FIG. 8 represents a result obtained by approximating a thinsolid brightness distribution using the Gaussian function. Then, thedensity estimation section 54 calculates the number of peaks indicatedby arrows A from the approximation result of the Gaussian function shownin FIG. 8. Further, the density estimation section 54 counts the numberof peaks per unit area to estimate the density (the degree ofconcentration) of the cells. That is, the density estimation section 54estimates the density of the cells by assuming that one peak in thebrightness distribution corresponds to one cell.

In the above description, the density of the cells on the single dotchain line shown in FIG. 6 is estimated, but a user may set and input asegment line for estimating the density using the input device 90.Further, a range for estimating the density may be designated in twodimensions using a rectangular range or a circular range, instead of thesegment.

Further, there is a case where a high NA captured image includes animage of a small amount of rubbish, or the like, in addition to cells.In a case where such a rubbish image is shown on a brightnessdistribution as a peak, it is not possible to calculate the density ofthe cells with high accuracy. FIG. 9 shows an example of a high NAcaptured image including such a rubbish image, in which a portionindicated by an arrow B in FIG. 9 corresponds to a rubbish image.

Further, in order to calculate the density of the cells with highaccuracy, it is preferable to remove the above-mentioned peak due to therubbish image. Specifically, in a case where the brightness distributionin a single dot chain line shown in FIG. 9 is acquired, a peak based onan image of rubbish or the like appears at a position spaced from a peakgroup based on an image of cells, as shown in FIG. 10. Thus, in a casewhere a peak indicated by an arrow in FIG. 10 is present, it ispreferable to calculate the number of peaks after the peak is removed asan isolation point due to the image of the rubbish or the like, andthen, to estimate the density of the cells.

The thickness of the subject S estimated by the thickness estimationsection 53 and the density of the subject S estimated by the densityestimation section 54 are output to the controller 51. The controller 51displays the input thickness and density of the subject S on the displaydevice 80 as a text, or outputs the result to another external device.In this embodiment, the controller 51 corresponds to an output sectionof the invention.

Returning to FIG. 1, the microscope control device 50 is connected tothe input device 90 and the display device 80. The input device 90 isprovided with an input device such as a keyboard or a mouse, andreceives a setting input from a user. Particularly, the input device 90in this embodiment receives setting inputs of a command formagnification change of the objective lens 31, a command for estimationof the thickness and density of the subject S, and the like.

The display device 80 is configured of a display device such as a liquidcrystal display, and displays a captured image (phase difference image)obtained by imaging in the imaging section 40, estimation results of thethickness and density of the subject S. or the like. Here, the displaydevice 80 may be configured using a touch panel, so that the displaydevice 80 may be used as the input device 90.

Next, an operation of the microscope image evaluation system of thisembodiment will be described with reference to a flowchart shown in FIG.11. Since the microscope image evaluation system of this embodiment hasa characteristic in estimating the thickness and density of the subjectS on the basis of a captured image obtained by imaging the subject S,this characteristic will be mainly described.

First, a command input for estimation of the thickness of the subject Sor estimation of the density of the subject S is input through the inputdevice 90 from a user (S10).

In a case where the command input for estimation of the thickness of thesubject S is input from the user, a low NA captured image is acquired bythe image acquisition section 52 (S12). The low NA captured image may beautomatically selected from captured images that are obtained by imagingat different magnifications and stored, or may be captured and acquiredat a low magnification by the phase difference microscope body when thecommand input for estimation of the thickness of the subject S isperformed from the user. Further, a plurality of captured images thatare obtained by imaging at different magnifications may be displayed onthe display device 80, and a low NA captured image obtained at a lowmagnification may be selected by the user using the input device 90.Here, in a case where the user selects a captured image having amagnification higher than a threshold value having a predeterminedmagnification, it is determined that the captured image is not suitablefor thickness estimation, and then, an alarm display or the like may beprovided. Further, when a plurality of captured images are displayed onthe display device 80, a mark may be provided to a captured image havinga magnification that is equal to or smaller than a threshold value of apredetermined magnification and suitable for thickness estimation.Further, a method for selecting two unspecified images from a pluralityof captured images obtained by imaging a subject under a condition inwhich the numerical aperture of the objective lens is changed, comparingimaging conditions of the two images, and determining a low NA capturedimage obtained under the condition in which the numerical aperture ofthe objective lens is relatively small and a high NA captured imageobtained captured under the condition in which the numerical aperture ofthe objective lens is relatively large may be used.

The low NA captured image acquired by the image acquisition section 52is input to the thickness estimation section 53. Further, for example,on the low NA captured image displayed on the display device 80, a rangefor estimating the thickness is designated by the user using the inputdevice 90 (S14).

The thickness estimation section 53 acquires a brightness distributionof the low NA captured image as described above on the basis of therange of estimation of the thickness input from the user (S16). Further,on the basis of the brightness distribution, the estimation of thethickness is performed with reference to the table as shown in FIG. 4(S18). The thickness of the subject S estimated by the thicknessestimation section 53 is displayed on the display device 80 under thecontrol of the controller 51 (S20).

Returning to S10, in a case where the command input for estimation ofthe density of the subject S is performed by the user, a high NAcaptured image is acquired by the image acquisition section 52 (S22).Similar to the low NA captured image, the high NA captured image may beautomatically selected from captured images that are obtained by imagingat different magnifications and stored in advance. Alternatively, whenthe command input for estimation of the thickness of the subject S isperformed by the user, the high NA captured image may be acquired byimaging at a high magnification by the phase difference microscope body.Further, a plurality of captured images that are obtained by imaging atdifferent magnifications may be displayed on the display device 80, anda high NA captured image obtained at a high magnification may beselected by the user using the input device 90. Here, in a case wherethe user selects a captured image having a magnification lower than athreshold value having a predetermined magnification, it is determinedthat the captured image is not suitable for density estimation, andthen, an alarm display or the like may be provided. Further, when aplurality of captured images are displayed on the display device 80, amark may be provided to a captured image having a magnification that isequal to or greater than a threshold value of a predeterminedmagnification and suitable for density estimation.

The high NA captured image acquired by the image acquisition section 52is input to the density estimation section 54. Further, for example, onthe high NA captured image displayed on the display device 80, a rangefor estimating the density is designated by the user using the inputdevice 90 (S24).

The density estimation section 54 acquires a brightness distribution ofthe high NA captured image as described above, on the basis of the rangeof estimation of the density input from the user (S26). Further, thebrightness distribution is approximated using the Gaussian function, andthen, the number of peaks is counted to perform the estimation of thedensity (S28). The density of the subject S estimated by the densityestimation section 54 is displayed on the display device 80 under thecontrol of the controller 51 (S30).

In the above description, the estimation of the thickness of the subjectS and the estimation of the density are selectively performed, but theinvention is not limited thereto, and both of the estimation of thethickness of the subject S and the estimation of the density may besimultaneously performed in parallel.

According to the microscope image evaluation system of this embodiment,a captured image obtained by imaging the subject S is acquired under thecondition in which the numerical aperture of the objective lens 31 ischanged, the thickness of the subject S is estimated on the basis of thelow NA captured image obtained under the condition in which thenumerical aperture of the objective lens 31 is relatively small, and thedensity of the subject S is estimated on the basis of the high NAcaptured image obtained under the condition in which the numericalaperture of the objective lens 31 is relatively large. Accordingly,since it is sufficient if two times of imaging of the low NA capturedimage and the high NA captured image can be performed, it is possible toevaluate the thickness and the density of the subject S in a shortimaging time.

Further, since it is sufficient if analysis of two captured images ofthe low NA captured image and the high NA captured image can beperformed, compared with a method for analyzing multiple sectionalimages in the related art, it is possible to reduce a load of theanalysis process. Further, as in the embodiment, in a case where theevaluation is performed using the brightness distribution, it ispossible to estimate the thickness and the density of the subjectthrough a more simplified process.

In the above-described embodiment, a captured image obtained at a lowmagnification is acquired as a low NA captured image, and a capturedimage obtained at a high magnification is acquired as a high NA capturedimage, but the invention is not limited thereto. For example, a capturedimage obtained using an aperture stop having a relatively small aperturemay be acquired as a low NA captured image, and a captured imageobtained using an aperture stop having a relatively large aperture maybe acquired as a high NA captured image.

FIG. 12 shows a configuration in which the above-described aperture stop14 is provided with respect to the phase difference microscope body ofthe microscope image evaluation system of this embodiment.

The aperture stop 14 is formed with an aperture 14 a through whichring-shaped illumination light passes. The size of the aperture 14 a maybe configured to be changeable. The change of the size of the aperture14 a may be manually performed, or may be automatically performed. Forexample, in a case where a command input for performing estimation ofthe thickness of the subject S is received from a user, the aperture 14a of the aperture stop 14 may be automatically changed to becomerelatively large. In a case where a command input for performingestimation of the density of the subject S is received from a user, theaperture 14 a of the aperture stop 14 may be automatically changed tobecome relatively small. The command input from the user may be receivedthrough the input device 90, or the size of the aperture 14 a forthickness estimation and the size of the aperture 14 a for densityestimation may be set in advance.

Further, a wavelength of illumination light of the phase differencemicroscope body may be configured to be changeable, and then, a capturedimage obtained with illumination light of a relatively long wavelengthmay be acquired as a low NA captured image, and a captured imageobtained with illumination light of a relatively short wavelength may beacquired as a high NA captured image. As a method for changing thewavelength of the illumination light, a light source having a differentwavelength may be configured to be manually or automatically changed, ora method for providing an optical filter to which illumination lightemitted from the light source is incident and manually or automaticallychanging the optical filter to change the wavelength of the illuminationlight may be used. As the wavelength of the illumination light incapturing the low NA captured image, for example, a wavelength of about780 nm may be used, and as the wavelength of the illumination light incapturing the high NA captured image, for example, a wavelength of about405 nm may be used.

In the above-described embodiment, the invention is applied to the phasedifference microscope, but the invention is not limited to the phasedifference microscope, and may be applied to other microscopes such as adifferential interference microscope or a bright field microscope.

EXPLANATION OF REFERENCES

-   -   10: illumination light emission section    -   11: white light source    -   12: slit plate    -   13: condenser lens    -   14: aperture stop    -   14 a: aperture    -   30: imaging optical system    -   31: objective lens    -   32: phase plate    -   33: image forming lens    -   40: imaging section    -   50: microscope control device    -   51: controller    -   52: image acquisition section    -   53: thickness estimation section    -   54: density estimation section    -   60: culture container    -   61: stage    -   62: stage driving section    -   80: display device    -   90: input device    -   S: subject

What is claimed is:
 1. A captured image evaluation apparatus comprising:a processor configured to acquire a plurality of captured imagesobtained by imaging a subject under conditions in which a numericalaperture (“NA”) of an objective lens is different; acquire a brightnessdistribution of a low NA captured image among the plurality of capturedimages; estimate a thickness of the subject on the basis of thebrightness distribution of the low NA captured image according to atable in which a brightness of the low NA captured image and thethickness of the subject are associated with each other; acquire abrightness distribution of a high NA captured image obtained under acondition in which the numerical aperture of the objective lens islarger than the numerical aperture of the objective lens of the low NAcaptured image, among the plurality of captured images, and estimate adensity of the subject by calculating a number of peaks per unit areaincluded in the brightness distribution of the high NA captured image;wherein a level of magnification sets a threshold, wherein the thicknessof the subject is estimated below the threshold, and wherein the densityof the subject is estimated above the threshold.
 2. The captured imageevaluation apparatus according to claim 1, wherein the processor isfurther configured to estimate the density of the subject on the basisof a shape of the brightness distribution.
 3. The captured imageevaluation apparatus according to claim 1, wherein the processor isfurther configured to approximate the brightness distribution using aGaussian function to calculate the peak.
 4. The captured imageevaluation apparatus according to claim 1, wherein the high NA capturedimage is a captured image obtained at a higher magnification than thelow NA captured image.
 5. The captured image evaluation apparatusaccording to claim 2, wherein the high NA captured image is a capturedimage obtained at a higher magnification than the low NA captured image.6. The captured image evaluation apparatus according to claim 1, whereinthe high NA captured image is a captured image obtained by illuminationlight of a shorter wavelength than the low NA captured image.
 7. Thecaptured image evaluation apparatus according to claim 2, wherein thehigh NA captured image is a captured image obtained by illuminationlight of a shorter wavelength than the low NA captured image.
 8. Thecaptured image evaluation apparatus according to claim 1, wherein thehigh NA captured image is a captured image obtained using an aperturestop having a larger aperture than the low NA captured image.
 9. Thecaptured image evaluation apparatus according to claim 1, wherein theprocessor is further configured to output the estimated thickness of thesubject and the estimated density of the subject.
 10. A captured imageevaluation method comprising: acquiring a plurality of captured imagesobtained by imaging a subject under conditions in which a numericalaperture (“NA”) of an objective lens is different; acquiring abrightness distribution of a low NA captured image among the pluralityof captured images; estimating a thickness of the subject on the basisof the brightness distribution of the low NA captured image according toa table in which a brightness of the low NA captured image and thethickness of the subject are associated with each other; acquiring abrightness distribution of a high NA captured image obtained under acondition in which the numerical aperture of the objective lens islarger than the numerical aperture of the objective lens of the low NAcaptured image, among the plurality of captured images, and estimating adensity of the subject by calculating a number of peaks per unit areaincluded in the brightness distribution of the high NA captured image;wherein a level of magnification sets a threshold, wherein the thicknessof the subject is estimated below the threshold, and wherein the densityof the subject is estimated above the threshold.
 11. A non-transitorycomputer recording medium storing a captured image evaluation programthat causes a computer to perform a method of, the method comprising:acquiring a plurality of captured images obtained by imaging a subjectunder conditions in which a numerical aperture (“NA”) of an objectivelens is different; acquiring a brightness distribution of a low NAcaptured image among the plurality of captured images; estimating athickness of the subject on the basis of the brightness distribution ofthe low NA captured image according to a table in which a brightness ofthe low NA captured image and the thickness of the subject areassociated with each other; acquiring a brightness distribution of ahigh NA captured image obtained under a condition in which the numericalaperture of the objective lens is larger than the numerical aperture ofthe objective lens of the low NA captured image, among the plurality ofcaptured images; and estimating a density of the subject by calculatinga number of peaks per unit area included in the brightness distributionof the high NA captured image; wherein a level of magnification sets athreshold, wherein the thickness of the subject is estimated below thethreshold, and wherein the density of the subject is estimated above thethreshold.